IL-2 is a soluble protein which is capable of modulating lymphocyte reactivity and promoting the long-term in vitro culture of antigen-specific effector T-lymphocytes (mitogenesis) and, in the past, has been produced by stimulating mouse, rat or human lymphocyte cells with a mitogen. For instance, Morgan et al. in Science 193:1007 (1976) and Ruscetti et al. in J. Immunol., 119:131 (1977), both discussed a process for culturing pooled normal human lymphocytes in Roswell Park Memorial Institute -1640 medium (hereinafter "RPMI-1640") containing autologous serum and the mitogen phytohemagglutinin (hereafter "PHA").
Gillis and Smith, in Nature, 268:154 (1977) reported preparing murine IL-2 by stimulating normal DBA/2 mouse spleen cells with the mitogen concanavalin A (hereafter "Con A") in an RPMI-1640 culture medium containing fetal calf serum (hereafter "FCS").
Farrar et al. in J. Immunol., 121:1353 (1978), also disclosed preparing IL-2 from murine spleen cells incubated with Con A in a tissue culture medium containing normal mouse serum (hereafter "NMS").
Gillis et al. reported generating IL-2 from murine and rat spleen cells cultured in a RPMI-1640 tissue culture medium supplemented with heat-inactivated FCS, penicillin-G, and gentamicin. The murine and rat spleen cells were stimulated by various mitogens including Con A, PHA, and pokeweed mitogen (hereafter "PKM"), J. Immunol., 120:2027 (1978).
IL-2 also has been prepared from human peripheral blood mononuclear cells by culturing the cells in RPMI-1640 medium supplemented with autologous human serum, penicillin, gentamicin, fresh L-glutamine, and PHA. Gillis et al. in J. Immunol., 124:1954 (1980).
Gillis et al. in J. Immunol., 125:2570 (1980), identified the preparation of IL-2 from T cell leukemia and lymphoma cell lines, specifically a radiation-induced splenic lymphoma from the B10.BR mouse (LBRM-33) cultured in RPMI-1640 supplemented with heat-inactivated FCS, 2.5.times.10.sup.-5 M 2-mercaptoethanol, N-2-hydroxy-piperazine-XI.sup.1 -2-ethene-sulfonic acid (hereafter "HEPES") buffer, penicillin, streptomycin and fresh L-glutamine. The cultures were stimulated with various mitogens including Con A and PHA.
IL-2 purified from these mouse, rat and human normal T-lymphocytes, has been found to retain different types of biological activity, including: (1) marked enhancement of thymocyte mitogenesis, Watson et al. J. Exp. Med., 150:849 (1979) and Gillis et al. supra, J. Immunol., 124:1954; (2) promotion of long-term in vitro proliferation of antigen specific helper or killer T cell lines, Gillis et al. supra, Nature, 268:154 and Watson, J. Exp. Med., 150:1510 (1979); and, (3) induction of cytotoxic T lymphocyte (hereafter "CTL") reactivity and plaque-forming cell responses in cultures of nude mouse spleen cells. Watson et al., supra, J. Exp. Med., 150:849 and Gillis et al., supra, J. Immunol., 124:1954. Accordingly, these identified biological activities of IL-2 indicate that IL-2 is useful in elevating immune responses and restoring immune deficient T cell populations (nude mouse spleen cells) to normal levels of cell and humoral immunity. Furthermore, these results suggest that IL-2 production and response are important parameters of immunological functions which may be useful in clinical diagnosis of aberrant immunity. Moreover, the fact that human IL-2 makes possible the in vitro proliferation of antigen specific human, mouse and rat killer T cells emphasizes the importance of human IL-2 as a research agent.
For these and similar reasons, human IL-2 is currently being evaluated as therapeutic in human clinical trials. IL-2 is a key regulator of immune function and also may be of value in the treatment of economic animals, particularly in cases of stress-induced immune deficiency of cattle. It has been repeatedly hypothesized that the stress that cattle undergo during shipment to and from grazing areas to feed lots causes elevation in steroid hormone production which leads to a diminution of immune responsiveness. When animals arrive at a feed lot, due to decreased immune reactivity, they fall prey to common bacterial and viral infections which, under normal situations, such animals have the ability to counteract. Various types of upper respiratory tract infections can occur. The effect of this "shipping fever" syndrome is that animals in such situations go off feed, lose weight, and can even die from these normally rejected disease-causing infectious agents. Gillis and colleagues several years ago showed that steroid hormones dramatically depressed immune responses via their capacity to diminish IL-2 production. In fact, in vitro addition of IL-2 to steroid-suppressed immune responses dramatically restored immune reactivity. Based on these results, it is not surprising to suggest that the use of bIL-2 in the treatment of shipping fever syndrome could result in elimination of the syndrome and restoration of the many millions of dollars which are lost as a result of shipping induced immune deficiencies. Unfortunately, methods for production of bIL-2 are not well characterized. As well, sources of natural bIL-2 have not proven to be a workable system for generating sufficient quantities of homogeneous IL-2 to thoroughly investigate its potential therapeutic usefulness.
One potential method of producing relatively large quantities of homogeneous bIL-2 is through recombinant DNA techniques. Recombinant DNA techniques have been developed for economically producing a desired protein once the gene coding for the protein has been isolated and identified. A discussion of such recombinant DNA techniques for protein production is set forth in the editorial and supporting papers in Volume 196 of Science (April, 1977). However, to take advantage of the recombinant DNA techniques discussed in this reference, the gene coding for bIL-2 must first be isolated.